Short CommunicationOpen Access cc icon

Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations

Jacob G Hodge

Jennifer L Robinson

Adam J Mellott

Jacob G Hodge

https://orcid.org/0000-0002-6165-3932

Bioengineering Graduate Program, University of Kansas, Lawrence, KS 66045, USA

Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA

Search for more papers by this author

Jennifer L Robinson

Bioengineering Graduate Program, University of Kansas, Lawrence, KS 66045, USA

Department of Chemical & Petroleum Engineering, University of Kansas, Lawrence, KS 66045, USA

Search for more papers by this author

Adam J Mellott

*Author for correspondence: Tel.: +1 913 588 8308;

E-mail Address: amellott@kumc.edu

Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA

Ronawk, LLC, Olathe, KS 66062, USA

Search for more papers by this author

Published Online:12 Oct 2022https://doi.org/10.2217/rme-2022-0140

View article

Abstract

Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, ‘stem-like’ MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of ‘stem-like’ MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.

Tweetable abstract

Novel tissue-mimetic 3D hydrogel system enhances the retention of nonsenescent ASC populations in vitro for up to 6 weeks in culture and eliminates the need to subculture, improving regenerative capacity of ASCs and their secreted biologics.

Keywords:

Papers of special note have been highlighted as: • of interest

References

1. Hass R, Kasper C, Bohm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): a comparison of adult and neonatal tissue-derived MSC. Cell Commun. Signal. 9, 12 (2011). • Review of mesenchymal stem/stromal cells (MSCs) and the different types of MSC populations, which also discusses the current and future role of MSC-derived regenerative therapies.
Medline CASGoogle Scholar
2. Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J. Mesenchymal stem cells for regenerative medicine. Cells 8(8), 886 (2019).
Medline CASGoogle Scholar
3. Pittenger MF, Discher DE, Peault BM, Phinney DG, Hare JM, Caplan AI. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen. Med. 4, 22 (2019).
MedlineGoogle Scholar
4. Musial-Wysocka A, Kot M, Majka M. The pros and cons of mesenchymal stem cell-based therapies. Cell Transplant. 28(7), 801–812 (2019).
MedlineGoogle Scholar
5. Lukomska B, Stanaszek L, Zuba-Surma E, Legosz P, Sarzynska S, Drela K. Challenges and controversies in human mesenchymal stem cell therapy. Stem Cells Int. 2019, 9628536 (2019).
MedlineGoogle Scholar
6. Koh J, Griffin DR, Archang MM et al. Enhanced in vivo delivery of stem cells using microporous annealed particle scaffolds. Small 15(39), e1903147 (2019).
MedlineGoogle Scholar
7. Choe G, Park J, Park H, Lee JY. Hydrogel biomaterials for stem cell microencapsulation. Polymers (Basel) 10(9), 997 (2018).
MedlineGoogle Scholar
8. Sivaraj D, Chen K, Chattopadhyay A et al. Hydrogel scaffolds to deliver cell therapies for wound healing. Front. Bioeng. Biotechnol. 9, 660145 (2021).
MedlineGoogle Scholar
9. Trounson A, McDonald C. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell 17(1), 11–22 (2015).
Medline CASGoogle Scholar
10. Walter MN, Wright KT, Fuller HR, Macneil S, Johnson WE. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp. Cell Res. 316(7), 1271–1281 (2010).
Medline CASGoogle Scholar
11. Watson SL, Marcal H, Sarris M, Di Girolamo N, Coroneo MT, Wakefield D. The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. Br. J. Ophthalmol. 94(8), 1067–1073 (2010).
Medline CASGoogle Scholar
12. Hu L, Wang J, Zhou X et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci. Rep. 6, 32993 (2016).
Medline CASGoogle Scholar
13. Eleuteri S, Fierabracci A. Insights into the secretome of mesenchymal stem cells and its potential applications. Int. J. Mol. Sci. 20(18), 4597 (2019).
Medline CASGoogle Scholar
14. Kabat M, Bobkov I, Kumar S, Grumet M. Trends in mesenchymal stem cell clinical trials 2004–2018: is efficacy optimal in a narrow dose range? Stem Cells Transl. Med. 9(1), 17–27 (2020).
Medline CASGoogle Scholar
15. Ahmed AS, Sheng MH, Wasnik S, Baylink DJ, Lau KW. Effect of aging on stem cells. World J. Exp. Med. 7(1), 1–10 (2017).
MedlineGoogle Scholar
16. Jiang T, Xu G, Wang Q et al. In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects. Cell Death Dis. 8(6), e2851 (2017).
Medline CASGoogle Scholar
17. Yin Q, Xu N, Xu D et al. Comparison of senescence-related changes between three- and two-dimensional cultured adipose-derived mesenchymal stem cells. Stem Cell Res. Ther. 11(1), 226 (2020).
Medline CASGoogle Scholar
18. Drela K, Stanaszek L, Nowakowski A, Kuczynska Z, Lukomska B. Experimental strategies of mesenchymal stem cell propagation: adverse events and potential risk of functional changes. Stem Cells Int. 2019, 7012692 (2019). • Overview of current and ongoing limitations with MSC-derived therapies, with one major limitation being the non-standardized ex vivo expansion of MSCs that is needed for production of large-scale cell numbers and the adverse effects associated with this process.
MedlineGoogle Scholar
19. Izadpanah R, Kaushal D, Kriedt C et al. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res. 68(11), 4229–4238 (2008).
Medline CASGoogle Scholar
20. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22(5), 675–682 (2004).
Medline CASGoogle Scholar
21. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 126(4), 677–689 (2006).
Medline CASGoogle Scholar
22. Dupont S. Role of YAP/TAZ in cell–matrix adhesion-mediated signalling and mechanotransduction. Exp. Cell Res. 343(1), 42–53 (2016).
Medline CASGoogle Scholar
23. Vining KH, Mooney DJ. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18(12), 728–742 (2017).
Medline CASGoogle Scholar
24. Ryu NE, Lee SH, Park H. Spheroid culture system methods and applications for mesenchymal stem cells. Cells 8(12), 1620 (2019).
Medline CASGoogle Scholar
25. Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev. Technol. 12(4), 207–218 (2014).
Medline CASGoogle Scholar
26. Zhou Y, Chen H, Li H, Wu Y. 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension. J. Cell. Mol. Med. 21(6), 1073–1084 (2017). • Demonstrates the potential beneficial role of utilizing softer 3D culture substrates, such as hydrogels, for improving the retention/expression of pluripotent stem-like genes in MSC populations.
Medline CASGoogle Scholar
27. Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J. Stem Cells 11(12), 1065–1083 (2019).
MedlineGoogle Scholar
28. Panchalingam KM, Jung S, Rosenberg L, Behie LA. Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review. Stem Cell Res. Ther. 6, 225 (2015).
MedlineGoogle Scholar
29. Kaminska A, Wedzinska A, Kot M, Sarnowska A. Effect of long-term 3D spheroid culture on WJ-MSC. Cells 10(4), 719 (2021).
Medline CASGoogle Scholar
30. Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103(4), 655–663 (2009).
Medline CASGoogle Scholar
31. Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture. Nat. Methods 13(5), 405–414 (2016). • Excellent review describing the fabrication, utilization and benefits of 3D hydrogels for use in cell culture and cell therapies.
Medline CASGoogle Scholar
32. Tsou YH, Khoneisser J, Huang PC, Xu X. Hydrogel as a bioactive material to regulate stem cell fate. Bioact. Mater. 1(1), 39–55 (2016).
MedlineGoogle Scholar
33. Moshayedi P, Nih LR, Llorente IL et al. Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain. Biomaterials 105, 145–155 (2016).
Medline CASGoogle Scholar
34. Chen J, Chin A, Almarza AJ, Taboas JM. Hydrogel to guide chondrogenesis versus osteogenesis of mesenchymal stem cells for fabrication of cartilaginous tissues. Biomed. Mater. 15(4), 045006 (2020).
Medline CASGoogle Scholar
35. Zigon-Branc S, Markovic M, Van Hoorick J et al. Impact of hydrogel stiffness on differentiation of human adipose-derived stem cell microspheroids. Tissue Eng. A 25(19–20), 1369–1380 (2019).
Medline CASGoogle Scholar
36. Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 7, 14 (2006).
MedlineGoogle Scholar
37. Chowdhury F, Li Y, Poh YC, Yokohama-Tamaki T, Wang N, Tanaka TS. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLOS ONE 5(12), e15655 (2010).
Medline CASGoogle Scholar
38. Gerardo H, Lima A, Carvalho J et al. Soft culture substrates favor stem-like cellular phenotype and facilitate reprogramming of human mesenchymal stem/stromal cells (hMSCs) through mechanotransduction. Sci. Rep. 9(1), 9086 (2019).
MedlineGoogle Scholar
39. Wei W, Ma Y, Yao X et al. Advanced hydrogels for the repair of cartilage defects and regeneration. Bioact. Mater. 6(4), 998–1011 (2021).
Medline CASGoogle Scholar
40. Bai R, Tian L, Li Y et al. Combining ECM hydrogels of cardiac bioactivity with stem cells of high cardiomyogenic potential for myocardial repair. Stem Cells Int. 2019, 6708435 (2019).
MedlineGoogle Scholar
41. Wang H, Shi J, Wang Y et al. Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction. Biomaterials 35(13), 3986–3998 (2014).
Medline CASGoogle Scholar
42. Distler T, Lauria I, Detsch R et al. Neuronal differentiation from induced pluripotent stem cell-derived neurospheres by the application of oxidized alginate–gelatin–laminin hydrogels. Biomedicines 9(3), 261 (2021).
Medline CASGoogle Scholar
43. Burnsed OA, Schwartz Z, Marchand KO, Hyzy SL, Olivares-Navarrete R, Boyan BD. Hydrogels derived from cartilage matrices promote induction of human mesenchymal stem cell chondrogenic differentiation. Acta Biomater. 43, 139–149 (2016).
Medline CASGoogle Scholar
44. Han WM, Mohiuddin M, Anderson SE, Garcia AJ, Jang YC. Co-delivery of Wnt7a and muscle stem cells using synthetic bioadhesive hydrogel enhances murine muscle regeneration and cell migration during engraftment. Acta Biomater. 94, 243–252 (2019).
Medline CASGoogle Scholar
45. Dimmeler S, Leri A. Aging and disease as modifiers of efficacy of cell therapy. Circ. Res. 102(11), 1319–1330 (2008).
Medline CASGoogle Scholar
46. Schimke MM, Marozin S, Lepperdinger G. Patient-specific age: the other side of the coin in advanced mesenchymal stem cell therapy. Front. Physiol. 6, 362 (2015).
MedlineGoogle Scholar
47. Rodier F, Campisi J. Four faces of cellular senescence. J. Cell Biol. 192(4), 547–556 (2011).
Medline CASGoogle Scholar
48. Van Deursen JM. The role of senescent cells in ageing. Nature 509(7501), 439–446 (2014).
Medline CASGoogle Scholar
49. Wagner W, Horn P, Castoldi M et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLOS ONE 3(5), e2213 (2008).
MedlineGoogle Scholar
50. Acosta JC, Banito A, Wuestefeld T et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat. Cell Biol. 15(8), 978–990 (2013).
Medline CASGoogle Scholar
51. Turinetto V, Vitale E, Giachino C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy. Int. J. Mol. Sci. 17(7), 1164 (2016).
MedlineGoogle Scholar
52. Ratushnyy A, Ezdakova M, Buravkova L. Secretome of senescent adipose-derived mesenchymal stem cells negatively regulates angiogenesis. Int. J. Mol. Sci. 21(5), 1802 (2020). • One of many articles highlighting some of the negative effects of the secretome and paracrine signaling of MSC populations that have undergone senescence. The authors show how senescent MSC populations can impair angiogenesis.
Medline CASGoogle Scholar
53. Liu J, Ding Y, Liu Z, Liang X. Senescence in Mesenchymal Stem Cells: Functional Alterations, Molecular Mechanisms, and Rejuvenation Strategies. Front. Cell. Dev. Biol. 8,258(2020).
MedlineGoogle Scholar
54. Turinetto V, Vitale E, Giachino C. Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy. Int. J. Mol. Sci. 17(7), 1164 (2016).
MedlineGoogle Scholar
55. Coppe JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5,99–118 (2010).
Medline CASGoogle Scholar

Vol. 18, No. 1

Supplemental Materials

Metrics

Downloaded 1,806 times

History

Received 8 August 2022

Accepted 22 September 2022

Published online 12 October 2022

Published in print January 2023

Information

Keywords

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/rme-2022-0140

Acknowledgments

Research reported in this publication was supported by KU startup funds (University of Kansas) and a MIRA grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM143081. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional private commercial resources, including donation of the T-Block hydrogels, was provided by Ronawk. Lastly, the authors would like to acknowledge the team at Nikon for providing the fluorescent confocal images in Figure 1C & Supplementary Videos via their AXR Confocal Imaging System.

Financial & competing interests disclosure

Research reported in this publication was supported by KU startup funds (University of Kansas) and a MIRA grant from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM143081. All authors declare that they do not have any conflict of interest except for A Mellott, who declares that they have financial interest with Ronawk, which is the company that produces and donated the T-Blocks used in this study. A Mellott is the co-founder and CEO of Ronawk. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Open access

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

PDF download